The fetus expresses paternally-derived as well as maternally- derived molecules and thus can be considered a semi-allogeneic graft with respect to the mother. In the human placenta, most classical MHC class I molecules are not expressed. However, a nonclassical MHC class I molecule, HLA-G, is highly expressed on fetal cells invading the maternal decidua. Although its function remains unclear, these data suggest that HLA-G plays a role in fetal-maternal interactions. Accumulating data suggest that HLA- G serves to protect the otherwise MHC class I-deficient fetal trophoblasts from lysis by maternal decidual natural killer (NK) cells by binding to inhibitory receptors expressed on these cells. Elucidation of the nature of HLA-G/NK cell interactions will be crucial in determining its importance in gestational disease. Aberrant HLA-G expression may lead to gestational complications such as pre-eclampsia, in which there is poor placentation, or spontaneous abortion. To test the hypothesis that HLA-G protects trophoblasts from NK cell lysis, we will generate HLA deletion mutants of the HLA-G-expressing choriocarcinoma (trophoblast) cell line JEG-3 using gene targeting techniques to knock out MHC class I genes. We will then determine whether these mutants are susceptible to lysis by NK cells. Furthermore, we will construct novel multivalent HLA-G tetramers for use as reagents so that we can systematically identify HLA-G receptors and the cells on which they are expressed.